Conceived and designed the experiments: JPS GMW TR. Performed the experiments: JPS GMW ILR NCG BB. Analyzed the data: JPS GMW ILR AY TR. Contributed reagents/materials/analysis tools: RTK. Wrote the paper: JPS TR.
The authors have declared that no competing interests exist.
Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet.
We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action.
The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity.
Steroid hormones are lipophilic signalling molecules, mediating a vast variety of physiological effects that depend on the cellular context of the target tissue. They act via steroid hormone receptors (SR), which belong to the nuclear receptor superfamily of ligand-activated transcription factors and serve as regulators of various target genes
Hormone binding and activity of SR is shaped by molecular chaperones
Both Hsp70 and Hsp90 feature a C-terminal EEVD motif that serves as acceptor site for cochaperones that harbour a tetratricopeptide repeat (TPR) domain
Many of the TPR proteins bring additional molecular functions to the SR-chaperone heterocomplexes. CHIP contains a C-terminal U-box that interacts with ubiquitin-conjugating enzymes and has been reported to promote degradation of various steroid receptors
PP5 is the only TPR-domain containing phosphatase identified so far; it has been shown to modulate a variety of cellular pathways
TPR2 is a J-domain containing cochaperone which has been demonstrated to modulate GR and PR signalling
Based on evidence from the literature and our own studies on the GR-inhibitory role of FKBP51, we had initiated a genotyping study that revealed a genetic association of this TPR protein with the response to medication in major depression
Since several TPR proteins should be able to compete with FKBP51 for binding to the binding site for TPR proteins in an Hsp90 dimer
To set up an assay for the determination of the influence of the seven selected TPR proteins CHIP, CYP40, FKBP51, FKBP52, PP5, TPR2 and XAP2 on the six steroid receptors GR, MR, PR, AR, ERα and ERβ, we established reporter gene assays for each of the receptors. For GR, MR, PR and AR, we made use of the hormone-responsive elements of the MMTV LTR promoter that was linked to the structural part of the firefly luciferase gene
GR and PR displayed the widest, AR a considerable, and MR and the two ERs a moderate range of hormone inducible activity in human neuronal SK-N-MC cells (
Neuronal SK-N-MC cells were transfected with a plasmid expressing one of the HA-tagged SRs, the MMTV firefly-luciferase reporter plasmid when transfecting GR, MR, PR, or AR, an ERE firefly-luciferase reporter plasmid for ERα and ERβ, and the Gaussia-KDEL control plasmid. After transfection, the cells were cultivated for 24 h in the presence of the indicated concentrations of hormone (DHT: Dihydrotestosterone) or EtOH as solvent control. Receptor activity represents firefly data normalized to Gaussia activities + S.E.M. of at least four independent experiments, each performed in duplicate.
Since the effects of FKBP51 on GR have been reported to be most pronounced at sub-saturating concentrations of hormone, we focused our further analyses on conditions that yielded significant, but not yet full activation of the respective steroid receptor. In addition, we also included one saturating concentration of hormone for each receptor.
To assess the effect of the TPR proteins on steroid receptor activity, each of the FLAG-tagged TPR proteins was co-expressed with each of the HA-tagged steroid receptors GR, MR, PR, AR, ERα, or ERβ, respectively, along with reporter and control plasmids. Since mammalian cells, in contrast to yeast, feature a number of different receptor-relevant TPR proteins, we reasoned that overexpression of a specific TPR protein is necessary to significantly enhance occupancy of the TPR acceptor site on Hsp90 by this specific cofactor. To test whether this is indeed the case under the conditions chosen we first evaluated the degree of overexpression for each of the TPR cofactors (
A, SK-N-MC cells were transfected with plasmid expressing one of the TPR proteins, lysed after 48 h and levels of the respective TPR protein was determined by Western blot analysis. B, HEK-293 cells were transfected with FLAG tagged Hsp90 along with FKBP52 expressing plasmid or control plasmid. Hsp90 was precipitated from lysates and the levels of co-precipitated cofactors were determined by Western blot.
Since our experimental design was further based on the assumption that selectively enhancing the level of one of the TPR cofactor results in changing the composition of the Hsp90 heterocomplexes, we tested this at the example of FKBP52 overexpression. Cells were transfected with FKBP52 expressing plasmid, and Hsp90 complexes were immunoprecipitated from cell lysates of FKBP52 overexpressing cells and control cells. While more FKBP52 was co-precipitated with Hsp90 complexes, all the other investigated TPR cofactors were less abundant (
The first observation we made in the reporter gene assays was that, in general, the changes in receptor activity upon co-expression of TPR cofactors were more pronounced for GR, MR, and PR than for AR, and even more than for the two ERs, which were almost not affected (
A-C, SK-N-MC cells in 96 well plates were transfected with the MMTV-Luc, Gaussia-KDEL control plasmid, a plasmid expressing one of the HA-tagged steroid hormone receptor (GR in A and B, MR in C and D) and constant amounts (200 ng) of a plasmid expressing one of the FLAG-tagged TPR-proteins. After transfection, the cells were cultivated for 24 h in the presence of hormone or vehicle as indicated. Relative receptor activity represents firefly data normalized to Gaussia activities and presented as relative stimulation to control + S.E.M. of at least four independent experiments performed in duplicate. Control cells were transfected with cloning plasmid instead of the TPR protein expressing plasmid. Lower panels of A and C, immunoblot of cell extracts, probed with anti-HA antibody visualizing steroid receptor expression, the same membrane probed with FLAG antibody demonstrating expression of TPR proteins and with actin antibody as loading control. D, After transfection, cells were cultivated in 0.1% or 10% SF-FCS containing media for 24 h in the presence of 0.03 nM fludrocortisol, or EtOH as vehicle control. Firefly luciferase data were normalized to Gaussia luciferase activities and are presented as relative stimulation + S.E.M. of three independent experiments performed in triplicate. * denotes
SK-N-MC cells were transfected with the MMTV-Luc (for PR and AR assays), or the ERE-Luc reporter plasmid (for ERα and ERβ assays), the Gaussia-KDEL control plasmid, a plasmid expressing the HA-tagged steroid hormone receptor as indicated and the plasmid expressing a FLAG-tagged TPR-protein. After transfection, cells were cultivated for 24 h in the presence of hormone as indicated. Relative receptor activity represents firefly data normalized to Gaussia activities and presented as relative stimulation to control + S.E.M. of at least four independent experiments performed in duplicate. Control cells were transfected with cloning plasmid replacing the TPR protein expression plasmid in the transfection mixture. Lower panels of A–D display immunoblots of cell extracts, probed with anti-HA antibody visualizing steroid receptor expression, the same membrane probed with FLAG antibody demonstrating expression of the TPR proteins and with actin antibody as loading control. * denotes
The TPR reactivity profile of MR was very similar, except for PP5 and XAP, which exerted only a marginally inhibitory effect on MR (
Similarly to GR, PR showed the highest activity when co-expressed together with CYP40 or FKBP52 (
We also monitored the expression levels of the co-expressed receptors and TPR proteins. There were some variations throughout the experiments, but overall there were no gross alterations in the levels of the steroid receptors in dependence of the co-expressed TPR cofactor, except for CHIP which often, albeit not consistently, led to lower receptor expression levels (
Since most of the TPR proteins had little impact on ERs' transcriptional activity, we wondered whether these two receptors are dependent on functional Hsp90 at all under our assay conditions. Therefore, we applied the specific Hsp90 inhibitor geldanamycin (GA), which has been shown to block Hsp90 activity by binding to its ATP pocket
SK-N-MC cells were transfected with 0.25 µg of one of the plasmids expressing ERα (A), ERβ (B) or GR (C), together with either ERE-Luc (A,B) or MMTV-Luc (C) as reporter plasmid and the Gaussia-KDEL control plasmid. After transfection, the cells were cultivated for 24 h in the presence of hormone and 10 ng/ml GA as indicated. Relative receptor activity represents Firefly data normalized to Gaussia activities and is presented as relative stimulation to control + S.E.M. of at least four independent experiments performed in duplicate. Lower panels, analysis of receptor expression after GA treatment in the presence or absence of hormone (10 nM estrogen, 500 nM cortisol).
It has been found that GA leads to degradation of Hsp90 client proteins such as GR
FKBP52, which does not change the activity of GR when co-expressed with this receptor in mammalian cells (
SK-N-MC cells were transfected with the MMTV-Luc reporter plasmid, the Gaussia-KDEL control plasmid, one of the plasmids expressing the HA-tagged GR or MR as indicated, and plasmids expressing FKBP51 and Cyp40 at the indicated amounts. After transfection, the cells were cultivated for 24 h in the presence of 10 nM cortisol (A) or 0.03 nM Fludrocortisol (B). Bar graphs indicate the relative reporter activity representing Firefly measurements normalized to Gaussia activities and presented as relative stimulation + S.E.M. of three independent experiments performed in triplicate. Lower panel of A displays immunoblots of cell extracts, probed with HA antibody demonstrating GR expression and the same membrane probed with FLAG antibody to detect overexpressed FKBP51 and Cyp40, and actin as control. In addition, antibodies directed against FKBP51 or Cyp40 were used to visualize the combined levels of endogenous and ectopic TPR protein.
The ability of TPR proteins to access heterocomplexes of steroid receptors and Hsp90 is assumed as prerequisite for their impact on these receptors. Therefore, we evaluated the relative incorporation of the TPR-proteins into steroid receptor complexes employing complementary co-immunoprecipitation. The estrogen receptors were not included, because they were only marginally affected by most of the TPR proteins. We expressed each of the HA-tagged steroid receptors in combination with each of the seven FLAG-tagged TPR proteins and performed co-immunoprecipitations with antibodies directed against the HA-tagged receptors or the FLAG-tagged TPR proteins, respectively, and visualized co-precipitated proteins by Westernblot analysis.
Since we observed varying efficiencies in the amount of precipitated protein using HA- or FLAG-directed antibodies, co-precipitated proteins were normalized to the precipitated primary target, and in case of HA-directed IPs also to the relative expression of the different TPR proteins.
For GR, the receptor IP revealed CHIP, FKBP51 and TPR2 as strong binders to the heterocomplex (
HEK-293 cells were transfected with 5 µg of a plasmid expressing HA-tagged GR together with 2-10 µg (to achieve similar expression levels) of one of the plasmids expressing a FLAG-tagged TPR protein. After 48-72 h cultivation in SF-FCS containing media, cells were harvested, lysed, and protein extracts prepared for immunoprecipitation of either the HA-tagged GR (A), or the FLAG-tagged TPR-proteins (B). A, Precipitation of HA-GR. Displayed is an example of an immunoblot that was probed with FLAG antibody to visualize co-precipitated TPR-proteins (upper right panel), and an immunoblot of the same membrane probed with HA antibody demonstrating precipitated GR (lower right panel). Left panel, quantification of the relative binding of the TPR-proteins to the steroid receptor heterocomplexes. FLAG- and HA-immunoblot signals of the eluates and FLAG immunoblot signals of the cell extracts, demonstrating expression of TPR proteins (C), were scanned and subjected to densitometry. The signal from the co-precipitated FLAG protein was corrected first by the amount of precipitated receptor and second by the amount of the TPR-protein present in the respective cell extract. Binding of TPR-proteins is presented relative to the mean of the normalized FLAG-eluate signals of CHIP, FKBP51, FKBP52, and PP5. Quantification represents the means of three independent experiments +S.E.M. B, precipitation of TPR proteins. Upper right panel, coomassie stained gel of eluates visualizing precipitated TPR-proteins (arrowheads) and co-precipitated Hsp90 and Hsp70. Lower right panel, immunoblots of eluates probed with HA antibody to demonstrate binding of GR to TPR-protein heterocomplexes. Left panel, quantification of the relative binding of co-precipitated proteins to the precipitated TPR-proteins. For quantification, signals were scanned and subjected to densitometry. Each HA immunoblot signal of the eluate was corrected by the amount of precipitated TPR-protein. Binding of steroid receptors is presented relative to the mean of the corrected HA eluate signals. Quantifications represent means of three independent experiments +S.E.M.
For MR, the interaction pattern of the TPR cofactors was similar to that of GR. Again, CHIP, FKBP51 and TPR2 exhibited strong interaction, while Cyp40 showed very little binding, both when immunoprecipitating the receptor or the cofactor (
HEK-293 cells were transfected as described for
In the case of PR, we observed the strongest interaction with the PR-Hsp90 heterocomplex for CHIP, FKBP51, and TPR2 (
HEK-293 cells were transfected as described for
Although AR showed less activity change in response to co-expression of TPR cofactors than GR, MR and PR, the TPR cofactors exhibited a distinct binding profile (
HEK-293 cells were transfected as described for
During maturation, the steroid receptor proceeds through a multi-chaperone machinery in which each step is characterized by a relative abundance of distinct chaperones
Since the FLAG-tagged proteins were precipitated with different efficiencies (although amounts of plasmids were adjusted so that the TPR proteins were expressed at similar levels, compare
Hsp90 interaction was detected for all TPR cofactors investigated here, as expected. However, there was a considerable difference in the relative amount of co-precipitated Hsp90 (
HEK cells were transfected and TPR cofactors immunoprecipitated as described in the legends to
Hsp70 binding was detected for CHIP and TPR2, as reported previously
The experiments described so far were based on increasing the abundance of a specific TPR cofactor in Hsp90 heterocomplexes. Considering the plethora of TPR cofactors in the cell, we pondered on the ability of mammalian cells to compensate for the loss of one of the proteins. Based on the inability of enhanced FKBP52 to significantly increase GR function, we reasoned that loss of an inhibitory factor, for example FKBP51, would have little effect. Therefore, we experimentally addressed the effect of loss of the established positive GR regulator FKBP52. Since our attempts to reduce FKBP52 using si-RNA resulted in only partial reduction (data not shown), we used mouse embryonic fibroblast (MEF) FKBP52 KO and WT cells. We found that stimulation of GR activity at saturating concentrations of hormone was not significantly affected (data not shown). However, higher concentrations of hormone were needed in FKBP52 ko cells to elicit a GR response comparable to that in WT MEF cells (
FKBP52-KO MEF cells (open symbols) or WT MEF cells (closed circles) were transfected with the MMTV-Luc reporter plasmid, the Gaussia-KDEL control plasmid, a plasmid expressing the HA-tagged mGR and either a plasmid expressing FLAG-tagged FKBP52 (+ect.52) or empty vector. After transfection, cells were cultivated for 24 h in the presence of hormone. Relative receptor activity represents firefly data normalized to Gaussia activities and is presented relative to the activity at saturating 300 nM corticosterone +S.E.M. of three independent experiments, each performed in triplicates. Significance of different receptor activation between FKBP52 KO cells and FKBP52 KO cells ectopically expressing FLAG-tagged FKBP52 was evaluated by one sampled T-test (* denotes
Since cells derived from different animals and cultivated for several generations can differ in numerous factors, it was mandatory to test whether the difference in the cortisol responsiveness between WT and FKBP52 KO MEF cells was indeed due to loss of FKBP52. Therefore, we overexpressed FKBP52 in FKBP52 KO MEF cells, which rendered the cortisol responsiveness indistinguishable from that of WT MEF cells (
How are molecular chaperones able to assist correct folding of a plethora of structurally divergent proteins? In general, the various chaperone factors protect non-native protein chains from misfolding and aggregation, but do not contribute conformational information to the folding process
Of the six steroid receptors, the closely homologous GR, MR and PR exhibited the strongest reaction to changes in the TPR-protein make-up of the cell (
Our study also documents numerous differences in the efficacies of the TPR proteins' influence on SR. Cyp40 exhibited only a minor effect on AR and PR, and no effect on GR, MR and the ERs, which concurs with its small binding affinity to Hsp90 and steroid receptor heterocomplexes in comparison to other TPR proteins (
CHIP efficiently inhibited the transactivational activity of GR, MR, PR, and moderately affected AR. It has been reported that CHIP induces degradation of GR, AR and ERα
Increasing or reducing the levels of TPR2 has been shown to reduce the activity of GR and PR
For XAP2, a moderate interaction with Hsp90 has been found before
FKBP51 and FKBP52 are the most intensely investigated TPR cofactors of steroid receptors. In particular for GR, important insight was gained from experiments in yeast, that characterised FKBP52 as stimulatory GR cofactor, while FKBP51 had no effect
In the study presented here, FKBP51 and FKBP52 exhibited divergent effects on the transcriptional activities of GR, MR, PR and AR. Consistent with a previous report on GR
Our experiments revealed an inhibitory effect of PP5 that was most pronounced in the case of GR. Previous studies examining the effect of PP5 on GR produced partly inconsistent results. Expression of the TPR domain of PP5 in CV-1 cells abolished GR-dependent transcription
The effects of the TPR proteins on SR observed here were significantly attenuated by saturating concentrations of hormone. This is consistent with an effect on hormone binding affinity. Different laboratories including ours provided evidence that later steps in steroid signal transduction are also affected by TPR cofactors, for example nuclear translocation
The MMTV-Luc reporter plasmid has been described previously
Mouse embryonic fibroblasts (Marc Cox and David Smith, Mayo Clinic Scottsdale, Arizona, USA), human neuroblastoma SK-N-MC (ATTC HTB-10) and HEK-293 (ATTC CRL-1573) cells were cultured under conditions described previously
To check expression of receptors and TPR-proteins replicate lysates were pooled, briefly sonicated and cleared by centrifugation. Alternatively receptors and TPR-proteins were coexpressed in 6 well plates with the same receptor to TPR-protein ratios as for the 96 well plates. To this end, SK-N-MC cells were seeded in 6 well plates (500,000 cells/well) in medium containing 10% steroid-free serum and cultured for 24 before transfection of 0.25 µg HA-tagged steroid hormone receptors and corresponding amounts of plasmids expressing TPR-proteins per well using ExGen (Fermentas) as described by the manufacturer. If needed, empty expression vector was added to the reaction to equal the total amount of plasmid in all transfections. Cells were cultured as for the reporter gene assay and lysed in buffer containing 20 mM Tris-HCl pH 6.8, 0.67% SDS, 3.3% Sacharose completed with Protease Inhibitor Cocktail (Sigma), briefly sonicated and cleared by centrifugation. Lysates were analyzed by SDS-PAGE followed by immunoblot.
To improve our understanding of the effects of various TPR proteins on steroid receptor mediated gene transcription, we performed one sample t-tests to evaluate the significance of difference of the hormone-stimulated activity of the receptor in the presence versus absence of coexpressed TPR protein. Significance values were corrected according to the Bonferroni procedure. The most pronounced differences with a significance level of p≤0.001 are labelled in
Immunoblot detection of proteins was performed largely as described
For immunoprecipitation of FLAG-tagged TPR proteins or HA-tagged steroid receptors, HEK-293 cells were transfected with 2–10 µg of a plasmid expressing a TPR protein (amounts were adjusted to ensure comparable expression levels) and 5 µg of a plasmid expressing a steroid receptor. For Hsp90 precipitations, 10 µg of FLAG-tagged Hsp90 expression plasmid were transfected together with 10 µg of FKBP52 plasmid. HEK 293 cells were chosen, because they efficiently expressed the proteins and showed the same results in reporter gene assays as SK-N-MC cells. Transfection was performed by electroporation of one confluent 10 cm (60 cm2) dish (∼5×106 cells) using a GenePulser (Bio-Rad, USA) at 350 V/700 µF in 400 µl of electroporation buffer (50 mM K2HPO4/KH2PO4, 20 mM KAc, pH 7.35, 25 mM MgSO4). Electroporated cells were replated in fresh medium containing 10% steroid-free serum containing medium and cultured for 3 days. Cells were harvested in cold PBS and lysed by resuspension in Lysis-Buffer A′ (130 mM NaCl, 20 mM Na2MoO4, 1 mM EDTA, 20 mM Tris-HCl pH 7.5, 10% Glycerol, 0.5% Triton X-100, completed with Protease Inhibtor cocktail, Sigma) for FLAG-TPR protein and receptor HA-IP, or in Hsp90 Lysis Buffer (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 20 mM Na2MoO4, 1 mM EDTA, 1 mM EGTA, 0.1% NP-40, 10% Glycerol, 0.5 mM DTT, completed with Protease Inhibitor cocktail, Sigma and Phosphatase Inhibitor cocktail, Roche) for the Hsp90 FLAG-IPs, followed by brief sonication (Branson Cell Disruptor B15, 3×5 s, output 3) and incubation on ice for 1 h. The lysate was cleared by centrifugation (10 min, 25.000 rcf, 4°C) and the protein concentration was determined. 1–2 mg of lysate was incubated overnight at 4°C with the anti-FLAG M2 agarose affinity resin (Sigma) or with anti-HA agarose affinity resin (Sigma), respectively. FLAG-beads (30 µl slurry) were treated as recommended by the manufacturer. The next day, the beads were washed 3 times with Lysis Buffer without detergent and samples were eluted with 70 µl of 1× FLAG-peptide solution (Sigma, 100–200 µg/ml) or HA-peptide solution (Sigma, 100 µg/ml), respectively, in 1× Tris-buffered saline (150 mM NaCl, 10 mM Tris-HCl pH 7.0).
For analysis of the (co)precipitated proteins, 5–15 µg of the cell lysates or 25 µl of the immunoprecipitates were separated by SDS-PAGE under denaturing conditions. Coomassie staining was used for detection of immunoprecipitated TPR-proteins and coimmunoprecipitated Hsp90 and Hsp70 in the FLAG-IP. For all other detections immunoblots were used, i.e. the (co)precipitated steroid receptors in the FLAG- and HA-IP, co-precipitated FLAG tagged TPR-proteins in the HA-IP, and p23 in the FLAG-IP. To analyze relative binding, the signals were subjected to densitometry. The coomassie stained gels or films were scanned at 16 bit with a calibrated densitometer (GS800, Bio-Rad, USA) and analyzed with the Kodak 1D Image Analysis software.
To calculate the relative binding of co-precipitated proteins we proceeded as follows: For relative binding of the receptors to the precipitated TPR-proteins (FLAG-IP) the HA-immunoblot signals of the eluates were first normalized with the Coomassie density signals of the precipitated TPR proteins. To be able to compare results between different experiments, we calculated these data to represent relative receptor binding among the TPR proteins. To this end, the normalized receptor (HA-IB) signal for each TPR protein was divided by the mean of the normalized receptor signals of all TPR proteins in each experiment. These ratios could then be averaged throughout the different experiments.
Conversely, to calculate the relative binding of the associated TPR proteins to the precipitated receptors (HA-IP), the FLAG-immunoblot signals of the HA-IP eluates were normalized first with the HA-immunoblot signals of the HA-IP eluates (to correct for variations in precipitation efficiencies of the receptors), and second to the FLAG-immunoblot signals of the lysate (to correct for differences in TPR protein expression). To calculate the mean of different experiments, like for the FLAG-IP, the normalized signals of each TPR protein were represented in reference to the relative binding of the other TPR proteins. Because of variabilities of the HA-IPs, the relative binding of each TPR protein were not normalized to the mean of all TPR proteins, but for each receptor to the mean of a subset of TPR proteins. The subset of TPR proteins used to calculate the mean binding in different experiments are displayed in the figure legends of each receptor.
To analyze the relative binding of Hsp90, the Hsp90 (FLAG-IP)- Coomassie signals were normalized to the Coomassie signals of the precipitated TPR proteins and this relative binding was used to calculate the mean binding in different experiments.
To analyze relative Hsp70 binding to TPR proteins (FLAG-IP), first the Hsp70 signal of the control reaction ( = background Hsp70 binding) was subtracted from the Hsp70 coomassie signals of each TPR protein, and these values were then normalized to the coomassie signals of the precipitated TPR proteins. Slightly negative values were considered as no binding and set to zero. This relative binding was used to calculate the mean binding in different experiments. To analyze significant binding of Hsp70 to FKBP51, a two tailed heteroscedastic students t-test were applied.
The authors thank Kathrin Hafner for outstanding technical assistance, Anja Kretzschmar for excellent support with MR reporter gene experiments, Cam Patterson, Anke Hoffmann, Christian Behl, Osborne Almeida, Len Neckers and Ulrich Hartl for kindly providing cDNAs and plasmids, David Smith and Marc Cox for kindly providing wt and FKBP52 KO MEF cells, Jürgen Zschocke for critical reading of the manuscript, and Florian Holsboer for continuous support.